Pet feeding habits and the microbiological contamination of dog food bowls: effect of feed type, cleaning method and bowl material.

BACKGROUND: Safe pet feeding practices and food bowl hygiene measures are important for minimising the risk of microbiological contaminations in the domestic environment. This study compares the practices reported by dog and cat caregivers, and investigates whether cleaning method, feed type or bowl material affects the microbiological contamination of dog food bowls. RESULTS: Data from 351 dog caregivers and 186 cat caregivers were collected via an online survey. The majority of dogs (70.7%) were fed twice daily, whereas cats (43%) were mostly fed ad libitum. The most common material for dog food bowls was metal (67.1%) versus plastic (38.1%) and metal (37.6%) for cats. Dog food bowls were most frequently cleaned after each meal (35.7%); whereas for cats, 21.5% were cleaned after each meal, 22.7% once a day and 19.3% 2-3 times a week. Total mesophilic aerobic bacteria counts (TMABc), Enterobacteriaceae counts and pathogenic bacteria (Salmonella spp., Campylobacter spp., Verotoxigenic E. coli [VTEC]) were assessed for 96 dog food bowls. TMABc were higher in metal vs. plastic bowls (p < 0.001) and in those used for wet food vs. dry food (p = 0.0397). Enterobacteriaceae counts were higher in bowls washed by hand vs. dishwasher (p = 0.0515), whereas no differences were found between hand washing vs. dry wiping. Salmonella spp., Campylobacter spp. or E. coli VTEC contaminations were not detected. CONCLUSIONS: The surveyed Italian dog and cat caregivers reported different habits concerning feeding frequency, food bowl material and cleaning frequency. Wet food and metal bowls were associated with higher levels of microbiological contamination of dog food bowls. Furthermore, in relation to wet washing methods, contaminations were likely to be greater following hand washing than they were following the use of a dishwasher. Practical guidelines for safe feeding practices and hygiene measures are needed to minimise the risk of microbiological contaminations in domestic environments.

A Fibre- vs. cereal grain-based diet: Which is better for horse welfare? Effects on intestinal permeability, muscle characteristics and oxidative status in horses reared for meat production.

Horses reared for meat production are fed high amounts of cereal grains in comparison with horses raised for other purposes. Such feeding practice may lead to risk of poor welfare consequences. The aim of this study was to investigate the effects of two feeding practices on selected metabolic parameters and production aspects. Nineteen Bardigiano horses, 14.3 ± 0.7 months of age, were randomly assigned to two groups-one fed with high amounts of cereal grains (HCG; n = 9; 43% hay plus 57% cereal grain-based pelleted feed) vs. one fed with high amounts of fibre (HFG; n = 10; 70% hay plus 30% pelleted fibrous feed)-for 129 days. At slaught on abattoir, biological and tissue samples were collected to evaluate the microbiological contamination of mesenteric lymph nodes and liver; selected meat quality traits (chemical composition and fatty acid profile of the Longissimus thoracis et lumborum muscle); and the oxidative status of the horse. A linear mixed model was used: dietary treatment and sex were fixed effects and their interaction analysed on production and metabolic parameters as dependent variables. Results showed an increased intestinal permeability in the horses fed HCG compared to HFG, according to the significant increased total mesophilic aerobic bacteria counts in mesenteric lymph nodes (p = 0.04) and liver samples (p = 0.05). Horses in HCG showed increased muscle pH (p = 0.02), lighter muscle colour (L) (p = 0.01), increased intramuscular fat concentrations (p = 0.03), increased muscle glutathione peroxidase and superoxide dismutase activities (p = 0.01 and p = 0.03, respectively). Moreover, horses in HCG had lower muscle water holding capacity at interaction with sex (p = 0.03, lower in female), lower muscle protein content (p = 0.01), lower concentration of muscle PUFAs (p = 0.05) and lower plasma catalase activities (p = 0.05). Our results showed that feeding a high cereal grains diet can have global effects on horse physiology, and thus represents a threat for their welfare.

Short communication: High-throughput sequencing approach to investigate Italian artisanal cheese production.

In this study, high-throughput sequencing (HTS) was used to investigate the microbiota of Robiola di Roccaverano production, an artisanal Protected Designation of Origin soft cheese made with raw goat milk by addition of a natural milk starter (NMS), from the Piedmont region of Italy. Different steps of production of Robiola di Roccaverano cheese at one artisanal dairy were monitored. Matched samples of milk, NMS, curd, and 5-d and 15-d matured cheeses were collected at different periods of the year. The DNA sequences obtained by HTS belonged to 5 phyla: Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Tenericutes. In milk, Proteobacteria and Firmicutes were mainly found, and several operational taxonomic units (OTU) belonging to contaminant bacteria such as Pseudomonas, Serratia, and Staphylococcus were observed. However, in NMS, curd, and 5- and 15-d cheeses, Firmicutes were principally observed where OTU of Lactococcus lactis were predominant, followed by Leuconostoc mesenteroides OTU. The results of the analysis showed high bacterial diversity in milk samples compared with NMS, curd, and 5- and 15-d cheeses, suggesting strong action of NMS in driving the characteristics of the final products.

Microbial characterization of an artisanal production of Robiola di Roccaverano cheese.

Robiola di Roccaverano, from the Piedmont region of Italy, is a Protected Designation of Origin soft cheese made with raw goat milk. The peculiarity of this cheese is that during the manufacturing process, a natural starter culture (NC) is added to raw milk. This study examined the viable microorganisms of technological interest, including lactic acid bacteria and fungal populations, in samples of raw milk, NC, and fresh and ripened cheese collected from one dairy using culture-dependent techniques. First, the isolated colonies were analyzed using random amplification of polymorphic DNA (RAPD) PCR, and strains with similar fingerprints were clustered together. Further, representative isolates of each group were subjected to 16S or 26S ribosomal DNA sequencing. Finally, species-specific PCR was conducted to distinguish the Lactococcus lactis ssp. lactis and Lc. lactis ssp. cremoris. Among the studied lactic acid bacteria, 13 RAPD profiles were obtained, corresponding to 9 different bacterial species or subspecies. Concerning mold and yeast isolates, 5 species were found that coincided with 5 RAPD types. Observing the strains isolated in the study, Lc. lactis was the most prevalent species in raw milk and NC samples, and Leuconostoc mesenteroides was the predominant species identified in 5- and 15-d cheese isolates. Furthermore, whereas only these 2 species were detected in NC, Enterococcus and Lactobacillus genera were found in raw milk and cheese, respectively. Concerning the mold and yeast isolates, in NC Kluyveromyces spp. was mainly found, and in cheese samples the representative species were Geotrichum candidum and Yarrowia lipolytica. Finally, raw milk and cheese safety were evaluated, and the samples complied with the standard required by European Commission regulation number 2073/2005.

Cross-contamination in canine and feline dietetic limited-antigen wet diets.

BACKGROUND: Adverse food reactions (AFRs) are defined as abnormal responses to an ingested food or food additive. Diagnosis and treatment of AFRs consist of the complete elimination of these ingredients in the dietary trial. Previous studies have demonstrated the presence of undeclared ingredients in commercial limited-antigen dry food diets that can compromise the results and efficacy of dietary elimination trails. The aim of this study was to assess a selection of commercial canine and feline dietetic limited-antigen wet foods for the potential cross-contamination of animal proteins from origins not mentioned on the label. RESULTS: Eleven canine and feline dietetic limited-antigen wet foods (9 novel animal protein foods, 1 vegetarian and 1 hydrolyzed) were analyzed by polymerase chain reaction (PCR) to detect the presence DNA of animal and vegetal origins. PCR analysis confirmed the contamination of 6 of the 11 (54.5%) limited-antigen wet diets with undeclared animal protein. One of these 6 diets was solely composed of animal protein sources completely unrelated to those declared on the label. None of the foods containing horse meat or fish were contaminated, and neither were the vegetarian or the hydrolyzed food products. Moreover, the results show that had zoological class primers only been used to check for cross-class contaminations, as are generally used in the pet food industry for in-house checks, the apparent contamination rate would have been significantly underestimated: less than 20% (3/11), instead of the actual rate of 54.7% using species-specific primers. CONCLUSION: This study reveals a high rate of cross-contamination in dietetic limited-antigen wet canine and feline foods, as previously described for dietetic dry limited-antigen foods (reported to be more than 80%). These results add new fuel to the discussion about the potential causes underlying the failure of elimination diets, since animal protein contaminants may actually be present in the commercial dietetic limited-antigen diets. AFRs may therefore occur as a result of inadequate practices in the pet food industry.

Isolation and characterisation of lactic acid bacteria from donkey milk.

During the last years the interest in donkey milk has increased significantly mainly because of its compelling functional elements. Even if the composition and nutritional properties of donkey milk are known, its microbiota is less studied. This Research Communication aimed to provide a comprehensive characterisation of the lactic acid bacteria in raw donkey milk. RAPD-PCR assay combined with 16S rDNA sequencing analysis were used to describe the microbial diversity of several donkey farms in the North West part of Italy. The more frequently detected species were: Lactobacillus paracasei, Lactococcus lactis and Carnobacterium maltaromaticum. Less abundant genera were Leuconostoc, Enterococcus and Streptococcus. The yeast Kluyveromyces marxianus was also isolated. The bacterial and biotype distribution notably diverged among the farms. Several of the found species, not previously detected in donkey milk, could have an important probiotic activity and biotechnological potential. This study represents an important insight to the ample diversity of the microorganisms present in the highly selective ecosystem of raw donkey milk.

Multiplex primer-extension assay for identification of Yersinia species.

A multiplex primer-extension reaction (PER) assay, was specifically designed for the identification of ten Yersinia species. The assay, directed towards the tufA (elongation factor Tu) gene, was tested on a total of 42 samples representing Yersinia species and non-Yersinia species. The primers used in the preliminary PCR, designed in highly conserved regions upstream and downstream of the diagnosis sites, successfully amplified a 587 bp fragment. The diagnosis sites were simultaneously interrogated using a multiplex PER and the results were confirmed by fragment sequencing. The proposed test provides an appropriate tool to monitor the presence of Yersinia spp. in food samples and to evaluate the potential hazard for consumers.

Degenerative myelopathy in German Shepherd Dog: comparison of two molecular assays for the identification of the SOD1:c.118G>A mutation.

Degenerative myelopathy (DM) is a late-onset, slowly progressive degeneration of spinal cord white matter which is reported primarily in large breed dogs. The missense mutation SOD1:c.118G>A is associated with this pathology in several dog breeds, including the German Shepherd Dog (GSD). The aims of the present study were to develop a tool for the rapid screening of the SOD1 mutation site in dogs and to evaluate the association of the polymorphism with DM in the German Shepherd breed. Two different techniques were compared: a minisequencing test and a real-time pcr allelic discrimination assay. Both approaches resulted effective and efficient. A sample of 47 dogs were examined. Ten subjects presented the symptoms of the illness; for one of them the diagnosis was confirmed by postmortem investigations and it resulted to be an A/A homozygote. In another clinically suspected dog, heterozygote A/G, the histopathological examination of the medulla showed moderate axon and myelin degenerative changes. GSD shows a frequency of the mutant allele equal to 0.17, quite high being a high-risk allele. Because canine DM has a late onset in adulthood and homozygous mutant dogs are likely as fertile as other genotypes, the natural selection is mild and the mutant allele may reach high frequencies. A diagnostic test, easy to implement, may contribute to control the gene diffusion in populations. The SOD1:c.118G>A mutation could be a useful marker for breeding strategies intending to reduce the incidence of DM.

Antibiotic resistance of Vibrio species isolated from Sparus aurata reared in Italian mariculture.

Extensive use of antimicrobial agents in finfish farming and the consequent selective pressure lead to the acquisition of antibiotic resistance in aquaculture environment bacteria. Vibrio genus represents one of the main pathogens affecting gilthead sea bream. The development of antibiotic resistance by Vibrio represents a potential threat to human health by exchange of resistant genes to human pathogens through food chain. The objective of the present study was to conduct a multisite survey on the antibiotic resistance of Vibrio spp. isolated from gilthead sea bream reared in Italian mariculture. Vibrio spp. strains were isolated from skin, gills, muscles and intestinal content of 240 gilthead sea bream. A random selection of 150 strains was sequenced for species identification. Resistance against 15 antimicrobial agents was tested by the broth microdilution method. Vibrio harveyi and Vibrio alginolyticus accounted for 36.7% and 33.3% of the isolates respectively. 96% of the strains showed multiple resistance to the tested drugs, with two strains, Vibrio aestuarianus and Vibrio harveyi resistant to 10 and 9 antibiotics, respectively. Ampicillin, amoxicillin, erythromycin and sulfadiazine showed low efficacy against Vibrio spp. Rational use of antimicrobial agents and surveillance on antibiotic administration may reduce the acquisition of resistance by microorganisms of aquatic ecosystems.

137Cesium (137Cs) assessment in wild boars from northwestern Italy.

Radionuclide contamination is a serious health issue caused by nuclear experiments and plant accidents, as seen for the Chernobyl and Fukushima nuclear plants. Italy has been especially interested in northwestern alpine regions, as have several other nations. The aim of this work was to indagate 134Cs and 137Cs contamination in wild boars, which were considered bioindicators sampled in the Chisone/Germanasca Valley and the Pellice Valley districts (Piedmont, Italy) in two hunting seasons (2014 and 2016). In the 2014 season, only the livers of the animals (n = 48) were sampled, whereas in 2016, five different anatomical sampling sites were sampled for each animal (n = 16). The analyses were conducted in an accredited laboratory (Agenzia Regionale per la Protezione dell'Ambiente-ARPA) by the aid of an HPGe detector (Ortec) with a relative efficiency of 50%. In general, the contamination levels registered in 2014 were under the detection limit for 134Cs and low for 137Cs (Chisone/Germanasca valley: min: 0.0, max: 23.9 median 11.0 Bq/kg vs Pellice valley: min 0, max: 31.7, median: 9.6 Bq/kg) and no health concern can be supposed. In the first-year samples, the liver showed a negative correlation between age and contamination level. In the second year of sampling, low levels were confirmed (min: 3.1 Bq/kg, max: 113.3; median 17.7 Bq/kg). Multiple sampling from the same animal showed that the diaphragm (median = 27.7 Bq/kg) kidney (27.4) and tongue (27.6) were more contaminated than the liver (17.7) and spleen (15.3). Moreover, a linear mixed model revealed a negative organ-by-age interaction, meaning that interorgan differences in contamination level were greater in younger (5-11 months) than in older (18-36 months) animals. Different feeding habits can be the explanation. Our paper shows that muscle sites (diaphragm and tongue) can be useful for radionuclide pollution surveillance in wild boar populations and that younger animals show more interorgan variability in contamination levels than older animals. More investigations are needed to confirm this correlation and to fulfill the request for more data to achieve better risk assessment.

Use of Biomolecular Tools to Control the Labels of Ethnic Food Coming from Eastern Europe.

In recent years, due to the large Romanian community present in Italy, the retail of foods coming from Eastern Europe has increased. The most common type of violation detected in these foods consists of incorrect labeling and species-replacement frauds. In this paper, the compliance of labels of 43 ethnic processed food coming from Eastern Europe and commercialized in Italy was evaluated by means of PCR and Sanger sequencing. Our data revealed 33% of non-compliant labels in samples containing swine, ruminants, and avian ingredients. These results demonstrate that PCR can be easily used for the identification of species in highly processed products, proving to be a rapid, effective, and economic method. On the other hand, samples reporting fish as ingredients highlighted the ineffectiveness of the applied sequencing protocol, due to the low informative property of targeted fragments or to the lack of consensus sequences in the case of uncommon species.

Ruminant rhombencephalitis-associated Listeria monocytogenes strains constitute a genetically homogeneous group related to human outbreak strains.

Listeriosis is a disease that causes significant economic losses at the farm level because of high morbidity and mortality in ruminants. This study was performed to investigate the role of ruminants in the epidemiology of listeriosis in northern Italy and the possible association of animal-adapted strains of Listeria monocytogenes with strains associated with human disease. Twenty ruminant rhombencephalitis isolates previously confirmed as L. monocytogenes by bacteriology and PCR were characterized by serotyping, pulsed-field gel electrophoresis, multi-virulence-locus sequence typing (MVLST), and multiplex single nucleotide polymorphism (mSNP) typing for the detection of epidemic clones. Subtyping results were subsequently compared with those obtained from human, food, and environmental isolates of L. monocytogenes, including 311 isolates from the University of Turin, Grugliasco, Italy, and 165 isolates representing major human listeriosis outbreaks worldwide, in addition to other unrelated isolates. Both mSNP typing and MVLST showed that 60% of the isolates analyzed belonged to epidemic clone I (ECI), which has been epidemiologically linked to several human outbreaks of listeriosis. In particular, the 1981 Canada outbreak was linked to the use of sheep manure and the 1985 California outbreak was linked to the use of raw cow's milk. In our study, ECI isolates were collected from different ruminant species on geographically and temporally distinct occasions for the last 13 years. Our results support the hypothesis that ruminants represent possible natural reservoirs of L. monocytogenes strains capable of causing epidemics of listeriosis in humans.

Evaluation of the biofilm-forming ability and molecular characterization of dairy Bacillus spp. isolates.

Food processing lines represents a suitable environment for bacterial biofilm formation. One of the most common biofilm-forming genera in dairy processing plants is Bacillus, which includes species that may have a negative impact on safety and/or quality of dairy products. In the current study, we evaluated the biofilm forming ability and molecular characteristics of dairy Bacillus spp. isolates (B. cereus and B. subtilis). Reference strains (B. cereus ATCC 14579 and B. subtilis NCTC 3610) were also included in the experiment. All isolates were screened by micro-titer plate (96 wells) to assess their ability to form biofilm. Then, they were tested on two common food contact surfaces (polystyrene and stainless steel) by using 6-well plates and AISI 316 stainless steel coupons. Biofilm formation, expressed as biofilm production index (BPI), was higher on polystyrene than stainless steel (except for B. cereus ATCC 14579). These observations were further confirmed by scanning electron microscopy, which allowed the microscopy observation of biofilm structure. Moreover, a possible correlation among total viable cell counts (CFU) and BPI was examined, as well as a connection among biofilm formation and bacterial cell hydrophobicity. Finally, whole genome sequencing was performed highlighting a genetic similarity among the strains belonging to the same species. The presence of selected genes involved in biofilm formation was also examined showing that strains with a greater presence of these genes were able to produce more biofilm in the tested materials. Additionally, for B. cereus strains enterotoxin genes were detected.

Novel multiplex single nucleotide polymorphism-based method for identifying epidemic clones of Listeria monocytogenes.

A novel primer extension-based, multiplex minisequencing assay targeting six highly informative single nucleotide polymorphisms (SNPs) in four virulence genes correctly identified and differentiated all four epidemic clones (ECs) of Listeria monocytogenes and 9 other strains initially misclassified as non-ECs. This assay allows rapid, accurate, and high-throughput screening for all known ECs of L. monocytogenes.

Mycobiota Composition of Robiola di Roccaverano Cheese along the Production Chain.

Robiola di Roccaverano is a Protected Designation of Origin (PDO) cheese from the Piedmont region of Italy. In this study, the mycobiota occurring during Robiola di Roccaverano production was elucidated. Samples of milk, Natural Milk Cultures (NMC), curd, 5- and 15-days ripened cheese were collected from one dairy plant and the mycobiota was analyzed by the metataxonomic approach. Milk samples showed a high diversity and Cladosporium, Kluyveromyces marxianus, Geotrichum candidum and Debaryomyces hansenii were found with higher relative abundance. This mycobiota remains quite stable in NMC and curd matrices although the relative abundance of K. marxianus and G. candidum yeasts increased significantly and shaped the fungal composition of 5- and 15-day ripened cheese.

The Bacterial and Fungal Microbiota of "Robiola di Roccaverano" Protected Designation of Origin Raw Milk Cheese.

Robiola di Roccaverano is an artisanal Protected Designation of Origin (PDO) soft cheese made with raw goat's milk and by the addition of Natural Milk Culture (NMC) to drive the fermentation process. Cheeses collected from five different dairy plants were analyzed for their bacterial and fungal microbiota diversity. Lactococcus lactis and Leuconostoc mesenteroides were the main bacterial population, while Galactomyces candidum and Kluyveromyces marxianus constituted the core mycobiota but many other minor taxa were observed, suggesting a high level of complexity in fungal composition by these cheeses compared to bacteria population.

Safety assessment of traditional Plaisentif cheese.

Traditional foods are gaining more and more market due to consumers' increasing willingness to buy products linked to national cultures: among these products, cheese plays an important role. Plaisentif is a traditional Piedmont cheese, only made during violets blooming season. The aim of this work is to evaluate the safety of this cheese, taking into account the EU Regulations. Microbiological hazards as well chemical, biogenic amines and mycotoxins, analysis were investigated. Salmonella spp. and Listeria monocytogenes were never detected in cheeses after ripening. Biogenic amines were present in very low quantities. Ochratoxin A was never detected and patulin was detected in over one cheese during the two years of sampling. This is the first attempt to characterize traditional Plaisentif cheese from a safety point of view. All the information acquired can be held as a necessary basis for reinforcing the culture of traditional products, for economic opportunities in mountainous regions and for safeguarding traditions and cultural identities.

Characterization of Vegetative Bacillus cereus and Bacillus subtilis Strains Isolated from Processed Cheese Products in an Italian Dairy Plant.

Processed cheese is a commercial product characterized by high microbiological stability and extended shelf life obtained through the application of severe heat treatment. However, spore-forming bacteria can survive through thermal processes. Among them, microorganisms belonging to Bacillus genus have been reported. In this study, we examined the microbiological population of the first hours' production of processed cheeses in an Italian dairy plant during two seasons, between June and October 2020. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify bacteria colonies, allowing the isolation of Bacillus cereus and Bacillussubtilis strains. These results were further confirmed by amplification and sequencing of 16 rRNA bacterial region. A multi-locus sequence type (MLST) analysis was performed to assess the genetic similarity among a selection of isolates. The fourteen B. cereus strains showed two sequence types: ST-32 was observed in only one strain and the ST-371 in the remaining thirteen isolates. On the contrary, all twenty-one B. subtlis strains, included in the study, showed a new allelic profile for the pycA gene, resulting in a new sequence type: ST-249. For B. cereus strains, analysis of toxin genes was performed. All isolates were positive for nheABC, entFM, and cytK, while hblABCD, bceT, and ces were not detected. Moreover, the biofilm-forming ability of B. cereus and B. subtilis strains was assessed, and all selected isolates proved to be biofilm formers (most of them were stronger producers). Considering the genetical similarity between isolates, jointly with the capacity to produce biofilm, the presence of a recurring Bacillus population could be hypothesized.

Development of a biomolecular assay for the identification of Listeria at species level.

In this article, we developed a biomolecular assay specifically designed for the identification of Listeria at species level. The proposed test was based on (i) a duplex PCR targeting a specific fragment for L. monocytogenes (plcA gene) and a common fragment for all the Listeria (16S rRNA gene) and (ii) a minisequencing of the common fragment to detect diagnostic sites for the differentiation of the other five species: L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, and L. grayi. The specificity of the assay was first tested on a total of 25 certified strains representing 6 Listeria species and 14 non-Listeria strains as negative control and validated on 124 wild isolates obtained from food samples. The proposed assay provides an appropriate tool for rapid identification of Listeria at species level and it should be of great benefit to the food industry as well as to regulatory or public health laboratories engaged in establishing the safety of food products and the management of listeriosis.

Development of a biomolecular assay for postmortem diagnosis of Taenia saginata Cysticercosis.

Bovine cysticercosis is caused by the larval stage of the human tapeworm Taenia saginata. According to European data on meat inspection, the prevalence ranges from 0.007% to 6.8%, but the real prevalence is considered to be at least 10 times higher. Laboratory confirmation of the etiological agent is based on gross, stereomicroscopic, and histological examination of submitted specimens. False identifications may occur, possibly because of death and degeneration of cysts, or because taeniid larvae and other tissue parasites, such as Sarcocystis spp., may cause similar macroscopic morphological lesions. Therefore, tests that can warrant sure identification of taeniid lesions and calcified cysts in the muscle are needed. The focus of our study was to develop a suitable postmortem test that could be applied on putative lesions by T. saginata cysticerci, as ambiguously diagnosed after routine meat inspection. In particular, we proposed a biomolecular assay targeting the mitochondrial cytochrome c oxidase subunit I gene (COI). For developing the polymerase chain reaction assay, viable cysts of Cysticercus bovis (n = 10) were used as positive reference samples, and those of Echinococcus granulosus (n = 3), Cysticercus tenuicollis (n = 3), and Sarcocystis spp. (n = 4) as reference negative controls. Further, to evaluate the applicability of the proposed assay, 171 samples of bovine muscular tissue, obtained from local slaughterhouses and containing lesions recognized as T. saginata cysticerci by macroscopic examination, were tested. The proposed test confirmed the diagnosis at postmortem inspection in 94.7% (162/171) of samples. In conclusion, the assay developed in this study, amplifying a short fragment from the mitochondrial gene COI, showed to be suitable for samples containing both viable and degenerating T. saginata cysticerci, yielding an unequivocal diagnosis.